Engineering α-amylase levels in wheat grain suggests a highly sophisticated level of carbohydrate regulation during development

Wheat starch degradation requires the synergistic action of different amylolytic enzymes. Our spatio-temporal study of wheat alpha-amylases throughout grain development shows that AMY3 is the most abundant isoform compared with the other known alpha-amylases. Endosperm-specific over-expression of AMY3 resulted in an increase of total alpha-amylase activity in harvested grains. Unexpectedly, increased activity did not have a significant impact on starch content or composition but led to an increase of soluble carbohydrate (mainly sucrose) in dry grain. In AMY3 overexpression lines (A3OE), germination was slightly delayed and triacylglycerol (TAG) content was increased in the endosperm of mature grain. Despite increased AMY3 transcript and protein content throughout grain development, alterations of alpha-amylase activity and starch granule degradation were not detected until grain maturation, suggesting a post-translational inhibition of alpha-amylase activity in the endosperm during the starch filling period. These findings show unexpected effects of a high level of alpha-amylase on grain development and composition, notably in carbon partitioning and TAG accumulation, and suggest the presence of a hitherto unknown regulatory pathway during grain filling.